EP1152778B1 - Hemocompatible surfaces and method for producing same - Google Patents

Hemocompatible surfaces and method for producing same Download PDF

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Publication number
EP1152778B1
EP1152778B1 EP00922483A EP00922483A EP1152778B1 EP 1152778 B1 EP1152778 B1 EP 1152778B1 EP 00922483 A EP00922483 A EP 00922483A EP 00922483 A EP00922483 A EP 00922483A EP 1152778 B1 EP1152778 B1 EP 1152778B1
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Prior art keywords
materials
hemocompatible
blood
cells
mixtures
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German (de)
French (fr)
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EP1152778A1 (en
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Michael Hoffmann
Roland Horres
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/08—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/18—Use of ingredients of undetermined constitution or reaction products thereof

Definitions

  • the present invention relates to hemocompatible surfaces that are characterized in that on and / or in the surface of Materials components of the outer layer of blood cells and / or Mesothelial cells are applied and / or introduced. Furthermore, the The present invention relates to a method for producing hemocompatible Surfaces and their use in wide areas of the Healthcare, medicine, dentistry, surgery, cosmetics and / or in areas directly related to blood, tissue and / or others Body fluids are in contact.
  • Blood clotting in vertebrates is a complex process in the event of an injury, shortly before life-threatening Protects blood loss.
  • the blood coagulation system is activated, among other things. through contact with non-physiological, ie "foreign" body Substances.
  • Substances that make the blood coagulation system active suppress are also referred to as anti-thrombogenic.
  • the activation of the blood coagulation system is a serious problem for the patient, particularly in the case of invasive interventions. This is particularly the case for those people who rely on implants, such as intracoronary stents, heart valves, prostheses, artificial vascular systems, dialyzers or oxygenators, catheters, biosensors and others are instructed. Contact with surgical sutures can also cause problems. So far, to prevent the formation of life-threatening vascular occlusions (thrombi), the blood coagulation system has been disabled or actively suppressed. This is usually done by administering anti-thrombogenic drugs, so-called anticoagulants.
  • DE 196 30 879 uses for coating substrates only chemically modified derivatives of polysaccharides.
  • the Disadvantages of this method are varied and range from an excessive preparative effort over multi-stage Synthesis steps, a wide range of undesirable Side reactions and poor yields all the way through poorer properties of the derivatives compared to commercial ones available anti-thrombogenic substances such as heparin.
  • Verhagen et al. (British Journal of Heamatology, 1996, 95: 542-549) describes the use of whole living cells of the endothelium or mesothelium for colonizing implants. Disadvantage of the Whole cell use here is that it is due to specific Cell surface proteins lead to immune reactions in patients rejection reactions to the coated implants to lead. Substances that trigger such an immune reaction are also called immunogenic. To avoid rejection by With this procedure, immune reactions must only be patient's own Cell material can be used. This is another disadvantage because growing these cells is very time and cost intensive. The use of whole cells is also problematic high shear forces to which these cells are exposed in the bloodstream. This leads to increased degradation of the cells on the surfaces, which can negatively affects the durability of the coated implants.
  • WO 93/01843, WO 95/29712 and DE 195 05 070 also describe the use of whole living endothelial cells for coating of non-physiological materials or the use of substances, which is the growth of living endothelial cells on artificial ones Favor materials.
  • the present invention has accordingly set itself the task blood or tissue compatible, i.e. hemocompatible surfaces for To make available that do not have the disadvantages mentioned above and at the same time for production on an industrial scale are suitable.
  • hemocompatible surfaces solved which are characterized in that they are artificial as materials and / or natural organic and / or inorganic compounds and / or mixtures thereof and / or materials used in invasive Interfering with blood and / or other body fluids in contact come and / or contain animal organs and / or organ parts and on and / or in the surface of these materials Oligosaccharide, polysaccharide and / or lipid portions of the Glycoproteins, glycolipids and / or proteoglycans from the outside Layer of blood cells and / or mesothelial cells applied and / or introduced are.
  • the hemocompatible surfaces according to the invention thus essentially mimic the outer surface of blood and / or mesothelial cells, equivalent to the imitation of the natural surface of non-thrombogenic cells and / or tissue.
  • the blood coagulation system is therefore neither activated nor actively suppressed by the hemocompatible surfaces.
  • blood coagulation which is triggered, for example, by secondary injuries (cuts or the like), can run completely naturally and undisturbed.
  • Another advantage of the present invention is that cells, such as platelets, do not adhere to the hemocompatible surfaces according to the invention. This is desirable according to the invention, since it minimizes the risk of thrombus formation, ie the risk of thrombosis (embolism) for the treated patient.
  • the hemocompatible surfaces according to the invention are free from side effects.
  • the hemocompatible surfaces are also distinguished in that they are non-thrombogenic in the long term. That your beneficial properties do not wear out over time, so as is the case with pharmaceutically active systems (e.g. Release system) is the case. Because of this, the Surfaces according to the invention also for continuous use, so that additional burdens and risks for the patient from repeated invasive interventions to renew the implants are minimized.
  • hemocompatible surfaces contain as Materials artificial and / or natural organic and / or inorganic compounds and / or mixtures thereof and / or Materials used in invasive blood and / or other procedures
  • Body fluids come into contact and / or animal organs and / or organ parts on and / or in their surface components of the outer layer of blood cells and / or mesothelial cells on and / or are introduced.
  • materials are all materials understand that are suitable according to the invention with cell components to be charged. This also includes all materials used in the invasive surgery or in the course of a corresponding aftercare Blood and / or body fluids can come into contact.
  • Organic compounds are, for example, synthetic manufactured or naturally occurring high molecular substances and to understand their derivatives. Examples include: all forms of Plastics, elastomers, silicones or fibrous materials. Which includes e.g. Polyethylene (PE), Polyvinylchloride (PVC), Polyurethane (PUR), Polyamides (PA), phenoplasts (PF), aminoplasts, polystyrene, polyester, Resins, silicones, rubbers, chemical fibers, cellulose fibers, Cellulose membranes, protein fiber materials, collagens and derivatives thereof or combinations thereof. Mixtures of these are also Polymers, so-called polymer blends according to the invention.
  • PE Polyethylene
  • PVC Polyvinylchloride
  • PUR Polyurethane
  • PA Polyamides
  • PF phenoplasts
  • aminoplasts aminoplasts
  • polystyrene polyester
  • Resins silicones
  • rubbers chemical fibers
  • cellulose fibers Cellulose membranes
  • protein fiber materials collagens and derivative
  • the hemocompatible surfaces according to the invention as Materials contain animal organs, organ parts or vascular systems. These can be heart valves and / or vascular systems, for example, pigs or cattle are particularly suitable as a source.
  • the hemocompatible surfaces according to the invention contain metals, metal oxides, alloys or ceramics, glasses and / or minerals as well as derivatives thereof or all conceivable combinations and / or mixtures thereof. According to the invention, all possible combinations of materials are conceivable.
  • the examples elaborate the present invention, but are not limitative.
  • the materials Components of the outer layer of blood cells and / or mesothelial cells one and / or applied.
  • the hemocompatible surfaces in and / or on the surface of materials contain glycoproteins, preferably glycophorins. These glycophorins are distinguished, inter alia, by non-thrombogenic properties and are therefore outstandingly suitable for producing hemocompatible surfaces according to the invention.
  • the glycophorins of the outer layer of erythrocytes determine, among other things, the blood group of a person. Analogously to the different blood groups A, B, AB and 0, the corresponding erythrocytes contain glycophorin A, glycophorin B or glycophorin 0 or corresponding mixtures thereof.
  • a possible immune response due to cross reactions of mutually incompatible blood groups, ie clumping of blood (coagulation) can easily be ruled out here by coordinating prior to an invasive procedure with regard to the blood group of the patient to be treated and the on and / or in the surface of materials applied and / or introduced glycophorins of the hemocompatible surfaces according to the invention intended for application.
  • Appropriate blood tests are common laboratory practice and are performed routinely. Provided that blood group compatibility is taken into account, hemocompatible surfaces containing glycophorin can therefore be used universally, ie not bound to a single patient.
  • the present invention relates to hemocompatible surfaces containing on and / or in the surface of the materials oligosaccharide, Polysaccharide and / or lipid portions of the glycoproteins, glycolipids and / or proteoglycans from the outer layer of blood cells and / or Mesothelial cells.
  • hemocompatible surfaces of the present Invention as oligosaccharide or polysaccharide portions of the Proteoglycans hyaluronic acids, chondroitin sulfates, dermatan sulfates, Contain heparan sulfates, keratin sulfates or mixtures thereof.
  • hemocompatible surfaces according to the invention are free of Side effects such as through chemically or pharmaceutically active Coatings are known.
  • the hemocompatible surfaces according to the invention are characterized by that they are also non-immunogenic. That means, they solve with the Patients do not have an immune response, increasing the risk of rejection the hemocompatible surfaces is minimized.
  • the hemocompatible surfaces are non-thrombogenic and / or non-immunogenic.
  • Another advantage is that due to the solid according to the invention Anchoring the non-thrombogenic components of the outer layer the blood and / or mesothelial cells on the materials almost none Degradation takes place on the hemocompatible surfaces. A This minimizes the risk of embolism from thrombosis. Furthermore, there is no accumulation of cells, e.g. Platelets the hemocompatible surfaces according to the invention. This minimizes also the risk of thrombosis.
  • the present invention also relates to a method for Production of the hemocompatible surfaces according to the invention, in which Glycophorins, oligosaccharide, polysaccharide and / or lipid portions of the Glycoproteins, glycolipids and / or proteoglycans from the outside Layer of blood cells and / or mesothelial cells are isolated and this Cell components through physical or chemical bonding and / or in the surface of materials made of artificial and / or natural organic and / or inorganic compounds and / or Mixtures of these and / or materials used in invasive interventions Blood and / or other body fluids come into contact and / or animal organs and / or organ parts applied and / or introduced become.
  • the components of the outer layer of Blood cells from whole blood and / or from cell fractions obtained therefrom isolated from human or animal origin This means that the Cell components from erythrocytes, leukocytes and / or thrombocytes or mixtures thereof can be isolated. Mixtures of are preferred Erythrocytes and leukocytes. Erythrocytes are particularly preferred.
  • the components from the outer layer of mesothelial cells are according to the invention from omentum, peritoneum and / or internal organs isolated
  • raw materials can be slaughterhouse waste.
  • Isolation of the components of the outer layer of blood cells, Mesothelial cells or tissue rich in mesothelial cells occurs in well-known way.
  • shredding extraction, filtration, Precipitation, gel filtration, ion exchange chromatography, Affinity chromatography, electrophoresis, enzymatic or chemical Degradation, drying, dissolution, dialysis, ultrafiltration etc.
  • the cell components for the application and / or introduction of the cell components a chemical on and / or in the surface of the materials Immobilization, photoimmobilization, adhesion, drying or a Combination of them performed. Covalent, ionic, incidental or electrostatic or adhesive bonds or Combinations thereof between the components of the outer layer of the cells and the surfaces of the materials.
  • the outer components are preferably applied or introduced Cell layer on / in the surface of materials by covalent Bonds.
  • a particular advantage of the present invention is that the manufacturing method according to the invention combines enormous economic improvements over previously known methods and consequently the hemocompatible surfaces according to the invention are suitable for manufacturing on an industrial scale. This is based, for example, on the fact that, according to the invention, cell components and no living cells are used, that no endogenous (endothelial) cells of the patient have to be used, that the starting material for isolating these cell components is inexpensive and available in large quantities (slaughterhouse waste), so that none Cell cultivation is required, which is very time and cost intensive.
  • Another advantage of the hemocompatible surfaces according to the invention is that they can be used universally and are not restricted to use for a single patient. This is a vital benefit for patients, especially in emergency operations.
  • the fields of application of the present invention are widely spread.
  • the present invention relates to the use of hemocompatible Surfaces in wide areas of the health sector, in medicine, Dentistry, surgery or cosmetics and / or in areas related to invasive procedures with blood, tissue and / or others Body fluids come into contact.
  • One liter of serum-free erythrocytes is 0.154 in 1 liter molar phosphate buffer pH 7 suspended and mixed with 1 U / ml papain. After 2 hours of incubation at 56 ° C for 20 minutes at 3000 g centrifuged and the supernatant then decanted. In this The supernatant is 100 ml of DEAE-Sepharose CL-6B ion exchange gel the company Pharmacia Biotech suspended. The gel so loaded will still be washed three times in 0.1 molar saline and into one Chromatography column filled. Elution is carried out using a linear Saline gradients in the range of 0.1 to 0.8 mol / l over a total elution volume of 2 liters.
  • the dialysate is made up to a volume of 100 ml and a concentration of 0.03 mol / l sodium acetate, 0.073 mol / l Tris (Tris (hydroxymethyl) aminomethane from Fluka) and pH 8.0, mixed with 1 U chondroitinase ABC and incubated for 15 hours at 37 ° C. After dialyzing against water and concentrating under a water jet vacuum the solution obtained is again on a column with 100 ml of DEAE-Sepharose CL-6B from Pharmacia Biotech. applied and how previously eluted.
  • the DMMB positive gradient fractions are dialyzed under a water jet vacuum to a volume of 1 ml concentrated and on a column for preparative gel filtration (60 cm x 2 cm) using a Sephacryl S-300 gel (Pharmacia Biotech.) Chromatograph. There are 60 fractions of 2 ml volume collected, detected with DMMB and the positive fractions pooled. After repeated dialysis and lyophilization, the purified is obtained Erythrocytenplasmamembran-heparan sulfate.
  • citrate blood is centrifuged at 3000 g in a centrifuge with a swing-out rotor and the supernatant plasma is suctioned off.
  • the cell sediment is mixed with 2 liters of 1% ammonium oxalate solution cooled to 4 ° C. and incubated for 30 minutes at the same temperature. After centrifugation at 500 g for 5 minutes, the red supernatant is discarded and the pellet is suspended in 2 liters of 1% ammonium oxalate solution cooled to 4 ° C., centrifuged at 500 g for 5 min and the washing process is repeated twice as described above.
  • the detergent extract is centrifuged at 10,000 g for 60 min, decanted and suspended in the supernatant and sedimented in 10 ml DEAE Sephadex A50 ion exchange gel from Pharmacia Biotech loaded gel is washed three more times in 0.1 molar sodium chloride solution and filled into a chromatography column, the column being eluted with a linear sodium chloride gradient of 0.1 to 0.8 mol / l over a total elution volume of 2 liters 100 fractions of 2 ml volume were collected and the fractions which gave a positive color reaction with dimethylmethylene blue (DMMB from Fluka) were combined The solution was concentrated at 26.7 hPa (20 torr) and 40 ° C. and dialyzed against water.
  • DMMB dimethylmethylene blue
  • the dialysate will adjusted to a volume of 100 ml and a concentration of 0.1 mmol / l calcium acetate and 0.1 mol / l sodium acetate, titrated to pH 7 with acetic acid, mixed with 1 U heparinase I, heparinase II and heparinase III and 15 hours incubated at 37 ° C. After dialyzing against water and concentrating under a water jet vacuum, the resulting solution is again placed on a column with 10 ml DEAE Sephadex A50 from Pharmacia Biotech. applied and eluted as described above.
  • the DMMB-positive gradient fractions are dialyzed, concentrated to a volume of 1 ml under water jet vacuum and on a column for preparative gel filtration (60 cm ⁇ 2 cm) using a Sepharose Cl-4B gel from Pharmacia Biotech. Chromatograph. 60 fractions of 2 ml volume are collected, detected with DMMB and the positive fractions are combined. After repeated dialysis and lyophilization, the purified leukocyte surface proteochondroitin sulfate is obtained.
  • a kilogram of fresh beef momentum is made with 0.9% NaCI solution washed, freeze-dried, ground and mixed with 1 liter of acetone Degreased stirring overnight at room temperature. After filtering off and drying the resulting powder in 6 molar urea solution suspended and stirred overnight at room temperature. To Centrifugation at 3000 g for 1 hour becomes the slimy supernatant decanted, chilled to 4 ° C, with the same volume 4 ° C cold 1 molar NaOH mixed and incubated for 15 hours at 4 ° C. After that neutralized with dilute HCI, dialyzed against water, at 1 hour Centrifuged 3000 g and the supernatant decanted.
  • 100 mg cellulose membrane are in a 2 percent solution of 3-aminopropyl-triethoxysilane placed in ethanol / water (50:50) and 24 Stirred at 45 ° C for hours. After that, the membranes with a lot Washed water and dried.
  • the membranes treated in this way are in a solution of 1 mg mesothelial cell surfaces Chondroitin sulfate in 80 ml 0.1 molar 2- (N-morpholino) ethanesulfonic acid buffer pH 4.75 submerged.
  • CME-CDI N-cyclohexyl-N'-2-morpholinoethyl carbodiimide methyl p-toluenesulfonate
  • a glass for example a cover slip for microscopy, is stirred in 5 ml of chromic sulfuric acid for 6 hours. Then it is washed with plenty of water, air-dried and heated to 50 ° C. in 15 ml of dioxane. Then 2.5 ml of a 2 molar N, N'-diisopropylethylamine solution in dioxane are added and the mixture is stirred for 30 min. Then 2.5 ml of a 1 molar CNCI solution in dioxane are added and the mixture is stirred for a further 2 hours. It is then washed first with dioxane, then with dioxane / water and finally with pure water.
  • the glass modified in this way is placed in 20 ml of a solution of 1 mol / l of ethylenediamine and 0.1 mol / l of NaHCO 3 , then heated to 50 ° C. and stirred at this temperature for 72 hours. Then 0.1 mg sphingoglycolipid from human erythrocytes is dissolved in 20 ml 0.1 molar NaHCO 3 and stirred together with the substituted glass at 60 ° C for 110 hours. Then 2.5 ml of ethanolamine are added and the mixture is stirred for a further 30 minutes. The coated glass is washed with 4 molar NaCl solution and then with plenty of water and air dried.
  • the metal workpiece is cleaned with hot water in an ultrasound bath for four hours, washed with acetone and degreased with chloroform in an Soxhlet extractor for one hour.
  • the workpiece cleaned in this way is dried and immersed in a 0.01-0.1 molar solution of ⁇ -hexadecenyltrichlorosilane in bicyclohexyl with stirring for 2-15 minutes, washed twice with chloroform and water and extracted with chloroform in a Soxhlet extractor for 15 minutes.
  • the workpiece is immersed in a solution of 2 ml of acetone and 100 mg of KmnO 4 in 18 ml of water at 0 ° C. for 45 minutes and passed through a CO 2 stream.
  • the workpiece prepared in this way is stirred for 90 minutes in a borate buffer solution (sodium tetraborate 0.065 mol / l, pH 9.5). Finally, it is stirred in a solution of 0.3 g of 4-azido-1-fluoro-2-nitrobenzene in one liter of ethanol at 37 ° C. overnight.
  • a borate buffer solution sodium tetraborate 0.065 mol / l, pH 9.5
  • 4-azido-1-fluoro-2-nitrobenzene in one liter of ethanol at 37 ° C. overnight.
  • 0.5 g of erythrocyte plasma membrane heparan sulfate is dissolved in one liter of 0.1 molar 2- (N-morpholino) ethanesulfonic acid (MES) buffer pH 4.75 and stirred with the workpiece at 4 ° C. for 48 hours.
  • MES N-morpholino
  • the erythrocyte plasma membrane heparan sulfate is covalently immobilized for 10 minutes by exposure to a high-pressure mercury lamp. After washing with 4 molar saline for 40 minutes, the workpiece is washed with water and then dried.
  • the membrane is gradually added Washed water, 1 mol / l soda solution, 1 mmol / l hydrochloric acid and water.
  • the amino cellulose thus obtained is soaked in a borate buffer solution for 90 minutes (Sodium tetraborate 0.065 molar, pH 9.5).
  • the membrane is in a solution of 0.3 g of 4-azido-1-fluoro-2-nitrobenzene stirred in a liter of ethanol at 37 ° C overnight.
  • Leukocyte surface chondroitin sulfate is 0.1 in one liter molar 2- (N-morpholino) ethanesulfonic acid buffer pH 4.75 dissolved and with 2.5 g of the azido cellulose prepared as described above at 4 ° C for Stirred for 48 hours.
  • the leukocyte surface chondroitin sulfate is covalently immobilized by exposure to a high-pressure mercury lamp for 10 minutes. After washing with 4 molar saline for 40 Minutes and water, the cellulose membrane is dried.
  • silicone foil 1 g is mixed with 20 ml of water and 2 ml of 3-aminopropyltriethoxysilane added and the pH adjusted to 3.5. Then it is heated to 75 ° C. for 2 hours, washed with water and dried.
  • the amino group-containing silicone thus obtained is with a 2.5 percent solution of glutardialdehyde in 0.05 molar Sodium phosphate buffer added and adjusted to pH 7. After 60 minutes The activated silicone thus produced is stirred with at room temperature a 0.1% solution of glycophorin A (Sigma) with stirring 2-4 Implemented for hours and washed with water.
  • iron (II) sulfate 100 ⁇ l concentrated sulfuric acid and 2 ml Methacrylic acid are dissolved in 250 ml of water. About this solution 125 mg sodium disulfite and 125 mg potassium peroxodisulfate added. Then this solution is two hours Room temperature through an annular 1 m long PVC hose from 3 mm inner diameter pumped. The expiring Graft polymerization is carried out by adding 100 mg of hvdroquinone canceled. Then the hose is washed thoroughly with water. A solution of 250 mg of CME-CDI (N-cyclohexyl-N'-2-morpholinoethyl) carbodiimide methyl cooled to 4 ° C.
  • CME-CDI N-cyclohexyl-N'-2-morpholinoethyl
  • p-toluenesulfonate in 250 ml 0.1 molar 2- (N-Morpholino) ethanesulfonic acid buffer pH 4.75 is at 4 ° C in 30 min Circle pumped through the hose.
  • the hose activated in this way is included 0.1 molar 2- (N-morpholino) ethanesulfonic acid buffer pH 4.75 washed.

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  • Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Hematology (AREA)
  • Surgery (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Materials For Medical Uses (AREA)
  • Silicates, Zeolites, And Molecular Sieves (AREA)
  • Saccharide Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Dental Preparations (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)

Abstract

The invention relates to hemocompatible surfaces, characterized in that they contain materials and that components of the outer layer of blood cells and/or mesothelial cells are deposited on and/or introduced into the surface of said materials. The invention also relates to a method for preparing hemocompatible surfaces and to their use in numerous areas of the health sector, such as medicine, dentistry, surgery, cosmetics or in fields relating to blood, tissue and/or other body fluids.

Description

Die vorliegende Erfindung betrifft hämokompatible Oberflächen, die sich dadurch auszeichnen, daß auf- und/oder in die Oberfläche von Werkstoffen Bestandteile der äußeren Schicht von Blutzellen und/oder Mesothelzellen auf- und/oder eingebracht sind. Ferner betrifft die vorliegende Erfindung ein Verfahren zur Herstellung hämokompatibler Oberflächen sowie deren Verwendung in weiten Bereichen des Gesundheitssektors, in der Medizin, Zahnmedizin, Chirurgie, Kosmetik und/oder in Bereichen, die direkt mit Blut, Gewebe und/oder anderen Körperflüssigkeiten in Kontakt stehen.The present invention relates to hemocompatible surfaces that are characterized in that on and / or in the surface of Materials components of the outer layer of blood cells and / or Mesothelial cells are applied and / or introduced. Furthermore, the The present invention relates to a method for producing hemocompatible Surfaces and their use in wide areas of the Healthcare, medicine, dentistry, surgery, cosmetics and / or in areas directly related to blood, tissue and / or others Body fluids are in contact.

Die Blutgerinnung stellt sich bei Vertebraten als ein komplexer Prozeß dar, der im Falle einer Verletzung kurzfristig vor lebensbedrohlichen Blutverlusten schützt. Aktiviert wird das Blutgerinnungssystem dabei u.a. durch den Kontakt mit unphysiologischen, also "körperfremden" Substanzen. Substanzen, die das Blutgerinnungssystem aktiv unterdrücken, werden auch als anti-thrombogen bezeichnet. Hingegen werden Substanzen, die das Blutgerinnungssystem gar nicht erst aktivieren als nicht-thrombogen definiert.Blood clotting in vertebrates is a complex process in the event of an injury, shortly before life-threatening Protects blood loss. The blood coagulation system is activated, among other things. through contact with non-physiological, ie "foreign" body Substances. Substances that make the blood coagulation system active suppress, are also referred to as anti-thrombogenic. On the other hand become substances that the blood coagulation system doesn't even have activate defined as non-thrombogenic.

Die Aktivierung des Blutgerinnungssytems stellt vor allem bei invasiven Eingriffen ein schwerwiegendes Problem für den Patienten dar. Dies ist insbesondere für diejenigen Menschen der Fall, die auf Implantate, wie beispielsweise Intracoronarstents, Herzklappen, Prothesen, künstliche Gefäßsysteme, Dialysatoren oder Oxygenatoren, Katheter, Biosensoren u.a. angewiesen sind. Auch der Kontakt mit chirurgischen Nahtmaterialien kann zu Problemen führen.
Bislang wird zur Vermeidung der Ausbildung lebensbedrohlicher Gefäßverschlüsse (Thromben) das Blutgerinnungssystem außer Kraft gesetzt oder aktiv unterdrückt. Dies erfolgt in der Regel durch die Gabe von anti-thrombogenen Medikamenten, sogenannten Antikoagulantien. Diese wiederum weisen eine Vielzahl starker Nebenwirkungen für den Patienten auf, wie beispielsweise Thrombozytopenie, Nausea, Erbrechen, Haarausfall, hämorrhagische Hautnekrosen, erhöhte Blutungsneigung etc.. Darüber hinaus ist im Falle der Verwendung von Intracoronarstents oder Herzklappen selbst die vollständige medikamentöse Unterdrückung der Blutgerinnung oftmals kein ausreichender Schutz vor der Ausbildung zum Teil tödlich wirkender Thrombosen.The activation of the blood coagulation system is a serious problem for the patient, particularly in the case of invasive interventions. This is particularly the case for those people who rely on implants, such as intracoronary stents, heart valves, prostheses, artificial vascular systems, dialyzers or oxygenators, catheters, biosensors and others are instructed. Contact with surgical sutures can also cause problems.
So far, to prevent the formation of life-threatening vascular occlusions (thrombi), the blood coagulation system has been disabled or actively suppressed. This is usually done by administering anti-thrombogenic drugs, so-called anticoagulants. These in turn have a large number of strong side effects for the patient, such as thrombocytopenia, nausea, vomiting, hair loss, hemorrhagic skin necrosis, increased bleeding tendency, etc. In addition, in the case of the use of intracoronar stents or heart valves, even complete drug suppression of the blood clotting is often not adequate protection against the formation of sometimes fatal thromboses.

Für weite Bereiche des Gesundheitssektors, der Medizin, Zahnmedizin, Chirurgie, Kosmetik oder im allgemeinen der Bereiche, die bei invasiven Eingriffen mit Blut und/oder anderen Körperflüssigkeiten in Kontakt stehen, ist daher die Vermeidung der zuvor genannten starken Nebenwirkungen durch Antikoagulantien von großer Bedeutung.For wide areas of the health sector, medicine, dentistry, Surgery, cosmetics or in general the areas involved in invasive Interfering with blood and / or other body fluids in contact stand, is therefore avoiding the aforementioned strong Side effects from anticoagulants of great importance.

Aus dem Stand der Technik sind verschiedene Verfahren bekannt, die unphysiologische "Fremdoberflächen" durch Beschichtung mit unterschiedlichen Substanzen blut- bzw. gewebeverträglicher (hämokompatibel) machen sollen.Various methods are known from the prior art which non-physiological "foreign surfaces" by coating with different substances more compatible with blood or tissue (hemocompatible).

So beschreibt die DE 28 31 360 ein Verfahren zur Beschichtung einer Oberfläche eines medizinischen Gegenstandes mit einer Substanz (Heparin), die das Gerinnungssystem aktiv unterdrückt, also anti-thrombogen ist. Jedoch weist diese Substanz den Nachteil erheblicher Nebenwirkungen für den Patienten auf, wie sie beispielhaft bereits zuvor aufgezählt wurden.DE 28 31 360 describes a method for coating a Surface of a medical object with a substance (Heparin), which actively suppresses the coagulation system, i.e. anti-thrombogenic is. However, this substance has the disadvantage more significant Side effects for the patient based on how they have been exemplified previously were enumerated.

In der DE 44 35 653 werden Materialien mit einer dünnen Lackschicht aus Polymeren, in die zusätzlich Arzneistoffe eingearbeitet sein können, beschichtet, wobei diese Lackschicht im Körper permanent degradiert und somit freigesetzt wird. Nachteile dieser Methode sind zum einen, daß durch den permanenten Zerfall der Beschichtung nur eine zeitlich begrenzte Wirkung möglich ist. Zum anderen ist durch die permanente Ablösung von Lackpartikeln die Gefahr der Ausbildung von Thrombosen, die zu Embolien führen können, sehr hoch.In DE 44 35 653 materials are made with a thin layer of lacquer Polymers into which additional drugs can be incorporated, coated, whereby this lacquer layer in the body is permanently degraded and is thus released. Disadvantages of this method are that due to the permanent disintegration of the coating only one time limited effect is possible. Second is the permanent Detachment of paint particles the risk of thrombosis formation, which can lead to embolism, very high.

Die DE 196 30 879 verwendet zur Beschichtung von Substraten ausschließlich chemisch modifizierte Derivate von Polysacchariden. Die Nachteile dieses Verfahrens sind dabei vielgestaltig und reichen von einem übermäßigen präparativen Aufwand über vielstufige Syntheseschritte, einem breiten Spektrum an unerwünschten Nebenreaktionen und mangelhaften Ausbeuten bis hin zu durchweg schlechteren Eigenschaften der Derivate verglichen mit kommerziell erhältlichen anti-thrombogenen Substanzen, wie beispielsweise Heparin.DE 196 30 879 uses for coating substrates only chemically modified derivatives of polysaccharides. The Disadvantages of this method are varied and range from an excessive preparative effort over multi-stage Synthesis steps, a wide range of undesirable Side reactions and poor yields all the way through poorer properties of the derivatives compared to commercial ones available anti-thrombogenic substances such as heparin.

Verhagen et al. (British Journal of Heamatology, 1996, 95: 542-549) beschreibt die Verwendung von ganzen lebenden Zellen des Endothels bzw. Mesothels zur Besiedlung von Implantaten. Nachteilig an der Verwendung ganzer Zellen ist hier, daß es aufgrund spezifischer Zelloberflächenproteine zu Immunreaktionen kommt, die bei den Patienten zu Abstoßungsreaktionen gegenüber den beschichteten Implantaten führen. Substanzen, die eine solche Immunreaktion auslösen, werden auch als immunogen bezeichnet. Zur Vermeidung einer Abstoßung durch Immunreaktionen muß bei diesem Verfahren ausschließlich Patienteneigenes Zellmaterial benutzt werden. Dies stellt einen weiteren Nachteil dar, da die Anzucht dieser Zellen sehr zeit- und kostenintensiv ist. Problematisch sind bei der Verwendung von ganzen Zellen ferner die hohen Scherkräfte, denen diese Zellen im Blutstrom ausgesetzt sind. Dies führt zu verstärkter Degradation der Zellen an den Oberflächen, was sich negativ auf die Haltbarkeit der beschichteten Implantate auswirkt. Verhagen et al. (British Journal of Heamatology, 1996, 95: 542-549) describes the use of whole living cells of the endothelium or mesothelium for colonizing implants. Disadvantage of the Whole cell use here is that it is due to specific Cell surface proteins lead to immune reactions in patients rejection reactions to the coated implants to lead. Substances that trigger such an immune reaction are also called immunogenic. To avoid rejection by With this procedure, immune reactions must only be patient's own Cell material can be used. This is another disadvantage because growing these cells is very time and cost intensive. The use of whole cells is also problematic high shear forces to which these cells are exposed in the bloodstream. This leads to increased degradation of the cells on the surfaces, which can negatively affects the durability of the coated implants.

Auch die WO 93/01843, WO 95/29712 und DE 195 05 070 beschreiben die Verwendung von ganzen lebenden Endothelzellen zur Beschichtung von unphysiologischen Materialien bzw. die Verwendung von Substanzen, die ein Anwachsen von lebenden Endothelzellen auf künstlichen Materialien begünstigen. Jedoch liegt auch hier allen Verfahren die Kultivierung lebender Endothelzellen zugrunde, verbunden mit den zuvor bereits beschriebenen Nachteilen hinsichtlich des Zeit- und Kostenaufwandes bzw. der erheblichen Einschränkung, daß das beschichtete Material nicht universell eingesetzt werden kann, sondern für jeden einzelnen Patienten gesondert hergestellt werden muß.WO 93/01843, WO 95/29712 and DE 195 05 070 also describe the use of whole living endothelial cells for coating of non-physiological materials or the use of substances, which is the growth of living endothelial cells on artificial ones Favor materials. However, here too all procedures lie Cultivation of living endothelial cells, associated with those previously disadvantages already described in terms of time and Cost expenditure or the considerable limitation that the coated material can not be used universally, but for each individual patient must be manufactured separately.

Aus der Patentschrift DE 36 39 561 ist die Herstellung von Substraten, die mit dem spezifischen Endothelzelloberflächen-Proteopolysaccharid HS-I beschichtet werden, bekannt. Nachteile dieses Verfahrens sind, daß auch hier zur Isolierung dieser Komponenten größere Mengen an Patienteneigenen Endothelzellen benötigt werden. Dies erfordert für jeden einzelnen Patienten eine zeit- und kostenintensive Kultivierung seiner körpereigenen Endothelzellen, an die sich zusätzlich eine aufwendige Präparation des Proteopolysaccharid HS-I anschließt. Aufgrund dessen ist eine großtechnische Herstellung von HS-I und somit eine wirtschaftliche Nutzung dieses Verfahrens zur Beschichtung von Implantaten nicht realisierbar.From the patent DE 36 39 561 is the manufacture of substrates that with the specific endothelial cell surface proteopolysaccharide HS-I be coated, known. Disadvantages of this method are that too here larger quantities of patient's own to isolate these components Endothelial cells are needed. This requires everyone individual patients a time and cost intensive cultivation of their the body's own endothelial cells, to which an additional complex Preparation of the proteopolysaccharide HS-I then. Because of that a large-scale production of HS-I and thus an economical one Do not use this method to coat implants realizable.

US-A-4 350 629 beschreibt hämokompatible Oberflächen welche Glucosaminoglykan beliebigen Ursprungs enthalten.US-A-4 350 629 describes hemocompatible surfaces which contain glucosaminoglycan of any origin.

Die hier vorliegende Erfindung hat sich demgemäß die Aufgabe gestellt, blut- bzw. gewebeverträgliche, also hämokompatible Oberflächen zur Verfügung zu stellen, die die zuvor genannten Nachteile nicht aufweisen und gleichzeitig für eine Herstellung im großtechnischen Maßstab geeignet sind. The present invention has accordingly set itself the task blood or tissue compatible, i.e. hemocompatible surfaces for To make available that do not have the disadvantages mentioned above and at the same time for production on an industrial scale are suitable.

Diese Aufgabe wird erfindungsgemäß durch hämokompatible Oberflächen gelöst, die sich dadurch auszeichnen, daß sie als Werkstoffe künstliche und/oder natürliche organische und/oder anorganische Verbindungen und/oder Mischungen davon und/oder Materialien, die bei invasiven Eingriffen mit Blut und/oder anderen Körperflüssigkeiten in Kontakt kommen und/oder tierische Organe und/oder Organteile enthalten und auf und/oder in die Oberfläche dieser Werkstoffe Oligosaccharid-Polysaccharid- und/oder Lipid-Anteile der Glykoproteine, Glykolipide und/oder Proteoglykane aus der äußeren Schicht von Blutzellen und/oder Mesothelzellen auf- und/oder eingebracht sind.This object is achieved by hemocompatible surfaces solved, which are characterized in that they are artificial as materials and / or natural organic and / or inorganic compounds and / or mixtures thereof and / or materials used in invasive Interfering with blood and / or other body fluids in contact come and / or contain animal organs and / or organ parts and on and / or in the surface of these materials Oligosaccharide, polysaccharide and / or lipid portions of the Glycoproteins, glycolipids and / or proteoglycans from the outside Layer of blood cells and / or mesothelial cells applied and / or introduced are.

Die erfindungsgemäß hämokompatiblen Oberflächen ahmen somit im wesentlichen die äußere Oberfläche von Blut- und/oder Mesothelzellen nach, gleichbedeutend mit der Imitation der natürlichen Oberfläche nichtthromogener Zellen und/oder Gewebe.
Das Blutgerinnungssystem wird folglich durch die hämokompatiblen Oberflächen weder aktiviert noch aktiv unterdrückt. Folglich kann eine Blutgerinnung, die z.B. durch sekundäre Verletzungen (Schnittwunden o.ä.) ausgelöst wird, vollkommen natürlich und ungestört ablaufen.
Ein weiterer Vorteil der vorliegenden Erfindung ist, daß eine Anhaftung von Zellen, wie beispielsweise Thrombocyten auf den erfindungsgemäß hämokompatiblen Oberflächen ausbleibt. Dies ist erfindungsgemäß erwünscht, da dadurch das Risiko der Ausbildung von Thromben, d.h. die Gefahr einer Thrombose (Embolie) für den behandelten Patienten minimiert ist. Die erfindungsgemäß hämokompatiblen Oberflächen sind frei von Nebenwirkungen.The hemocompatible surfaces according to the invention thus essentially mimic the outer surface of blood and / or mesothelial cells, equivalent to the imitation of the natural surface of non-thrombogenic cells and / or tissue.
The blood coagulation system is therefore neither activated nor actively suppressed by the hemocompatible surfaces. As a result, blood coagulation, which is triggered, for example, by secondary injuries (cuts or the like), can run completely naturally and undisturbed.
Another advantage of the present invention is that cells, such as platelets, do not adhere to the hemocompatible surfaces according to the invention. This is desirable according to the invention, since it minimizes the risk of thrombus formation, ie the risk of thrombosis (embolism) for the treated patient. The hemocompatible surfaces according to the invention are free from side effects.

Erfindungsgemäß zeichnen sich die hämokompatiblen Oberflächen ferner dadurch aus, daß sie langfristig nicht-thrombogen sind. D.h. ihre vorteilhaften Eigenschaften verbrauchen sich nicht im Laufe der Zeit, so wie es beispielsweise bei pharmazeutisch aktiven Systemen (z.B. Release-System) der Fall ist. Aufgrund dessen eignen sich die erfindungsgemäßen Oberflächen auch zum Dauereinsatz, so daß zusätzliche Belastungen und Risiken für die Patienten durch wiederholte invasive Eingriffe zur Erneuerung der Implantate minimiert werden.According to the invention, the hemocompatible surfaces are also distinguished in that they are non-thrombogenic in the long term. That your beneficial properties do not wear out over time, so as is the case with pharmaceutically active systems (e.g. Release system) is the case. Because of this, the Surfaces according to the invention also for continuous use, so that additional burdens and risks for the patient from repeated invasive interventions to renew the implants are minimized.

Erfindungsgemäß enthalten die hämokompatiblen Oberflächen als Werkstoffe künstliche und/oder natürliche organische und/oder anorganische Verbindungen und/oder Mischungen davon und/oder Materialien, die bei invasiven Eingriffen mit Blut und/oder anderen Körperflüssigkeiten in Kontakt kommen und/oder tierische Organe und/oder Organteile auf und/oder in deren Oberfläche Bestandteile der äußeren Schicht von Blutzellen und/oder Mesothelzellen auf- und/oder eingebracht sind.According to the hemocompatible surfaces contain as Materials artificial and / or natural organic and / or inorganic compounds and / or mixtures thereof and / or Materials used in invasive blood and / or other procedures Body fluids come into contact and / or animal organs and / or organ parts on and / or in their surface components of the outer layer of blood cells and / or mesothelial cells on and / or are introduced.

Unter Werkstoffen sind im Sinne der Erfindung sämtliche Materialien zu verstehen, die sich erfindungsgemäß dazu eignen mit Zellbestandteilen beaufschlagt zu werden. Ebenso zählen hierzu alle Materialien, die bei invasiven Eingriffen oder im Zuge einer entsprechenden Nachsorge mit Blut und/oder Körperflüssigkeiten in Kontakt kommen können.In the context of the invention, materials are all materials understand that are suitable according to the invention with cell components to be charged. This also includes all materials used in the invasive surgery or in the course of a corresponding aftercare Blood and / or body fluids can come into contact.

Unter organischen Verbindungen sind beispielsweise synthetisch hergestellte oder natürlich vorkommende hochmolekulare Stoffe und deren Derivate zu verstehen. Beispiele hierfür sind u.a. alle Formen von Kunststoffen, Elastomeren, Silikonen oder Faserstoffen. Hierzu zählen z.B. Polyethylene (PE), Polyvinylchloride (PVC), Polyurethane (PUR), Polyamide (PA), Phenoplaste (PF), Aminoplaste, Polystyrol, Polyester, Harze, Silikone, Kautschuke, Chemiefaserstoffe, Zellulosefaserstoffe, Zellulosemembranen, Proteinfaserstoffe, Collagene sowie Derivate davon oder Kombinationen davon. Ferner sind auch Mischungen dieser Polymere, sogenannte Polymerblends erfindungsgemäß umfaßt. Organic compounds are, for example, synthetic manufactured or naturally occurring high molecular substances and to understand their derivatives. Examples include: all forms of Plastics, elastomers, silicones or fibrous materials. Which includes e.g. Polyethylene (PE), Polyvinylchloride (PVC), Polyurethane (PUR), Polyamides (PA), phenoplasts (PF), aminoplasts, polystyrene, polyester, Resins, silicones, rubbers, chemical fibers, cellulose fibers, Cellulose membranes, protein fiber materials, collagens and derivatives thereof or combinations thereof. Mixtures of these are also Polymers, so-called polymer blends according to the invention.

In einer besonderen Ausführungsvariante der vorliegenden Erfindung können die erfindungsgemäß hämokompatiblen Oberflächen als Werkstoffe tierische Organe, Organteile oder Gefäßsysteme enthalten. Dies können beispielsweise Herzklappen und/oder Gefäßsysteme sein, wobei sich als Quelle Schweine oder Rinder besonders eignen.In a special embodiment variant of the present invention can the hemocompatible surfaces according to the invention as Materials contain animal organs, organ parts or vascular systems. These can be heart valves and / or vascular systems, for example, pigs or cattle are particularly suitable as a source.

Als Beispiele für anorganische Verbindungen enthalten die erfindungsgemäß hämokompatiblen Oberflächen Metalle, Metalloxide, Legierungen, oder Keramiken, Gläser und/oder Mineralien sowie Derivate davon oder alle denkbaren Kombinationen und/oder Mischungen davon.
Erfindungsgemäß sind alle Kombinationsmöglichkeiten von Werkstoffen denkbar. Die Beispiele führen die vorliegende Erfindung näher aus, sind jedoch nicht limitierend.As examples of inorganic compounds, the hemocompatible surfaces according to the invention contain metals, metal oxides, alloys or ceramics, glasses and / or minerals as well as derivatives thereof or all conceivable combinations and / or mixtures thereof.
According to the invention, all possible combinations of materials are conceivable. The examples elaborate the present invention, but are not limitative.

Erfindungsgemäß sind in und/oder auf der Oberfläche der Werkstoffe Bestandteile der äußeren Schicht von Blutzellen und/oder Mesothelzellen ein- und/oder aufgebracht.According to the invention are in and / or on the surface of the materials Components of the outer layer of blood cells and / or mesothelial cells one and / or applied.

In einer Ausführungsform der vorliegenden Erfindung enthalten die hämokompatiblen Oberflächen in und/oder auf der Oberfläche von Werkstoffen Glykoproteine, bevorzugt Glykophorine. Diese Glykophorine zeichnen sich u.a. durch nicht-thrombogene Eigenschaften aus und eignen sich dadurch hervorragend zur Herstellung erfindungsgemäß hämokompatibler Oberflächen.
Durch die Glykophorine der äußeren Schicht von Erythrocyten ist u.a. die Blutgruppenzugehörigkeit eines Menschen festgelegt. Analog zu den verschiedenen Blutgruppen A, B, AB und 0 enthalten die korrespondierenden Erythrocyten Glykophorin A, Glykophorin B oder Glykophorin 0 oder entsprechende Mischungen davon.
Eine mögliche Immunantwort durch Kreuzreaktionen nicht miteinander verträglicher Blutgruppen, d.h. ein Verklumpen von Blut (Koagulation) kann hier auf einfache Weise dadurch ausgeschlossen werden, daß vor einem invasiven Eingriff eine Abstimmung erfolgt, hinsichtlich der Blutgruppe des zu behandelnden Patienten und der auf und/oder in die Oberfläche von Werkstoffen auf- und/oder eingebrachten Glykophorine der zur Applikation beabsichtigten erfindungsgemäß hämokompatiblen Oberflächen. Entsprechende Bluttests sind gängige Laborpraxis und werden entsprechend routinemäßig durchgeführt. Unter der Voraussetzung der Beachtung der Blutgruppenverträglichkeit sind somit auch Glykophorin enthaltende hämokompatible Oberflächen universell einsetzbar, d.h. nicht an einen einzigen Patienten gebunden.In one embodiment of the present invention, the hemocompatible surfaces in and / or on the surface of materials contain glycoproteins, preferably glycophorins. These glycophorins are distinguished, inter alia, by non-thrombogenic properties and are therefore outstandingly suitable for producing hemocompatible surfaces according to the invention.
The glycophorins of the outer layer of erythrocytes determine, among other things, the blood group of a person. Analogously to the different blood groups A, B, AB and 0, the corresponding erythrocytes contain glycophorin A, glycophorin B or glycophorin 0 or corresponding mixtures thereof.
A possible immune response due to cross reactions of mutually incompatible blood groups, ie clumping of blood (coagulation) can easily be ruled out here by coordinating prior to an invasive procedure with regard to the blood group of the patient to be treated and the on and / or in the surface of materials applied and / or introduced glycophorins of the hemocompatible surfaces according to the invention intended for application. Appropriate blood tests are common laboratory practice and are performed routinely. Provided that blood group compatibility is taken into account, hemocompatible surfaces containing glycophorin can therefore be used universally, ie not bound to a single patient.

Die vorliegende Erfindung betrifft hämokompatible Oberflächen enthaltend auf und/oder in der Oberfläche der Werkstoffe Oligosaccharid-, Polysaccharid- und/oder Lipid-Anteile der Glykoproteine, Glykolipide und/oder Proteoglykane aus der äußeren Schicht von Blutzellen und/oder Mesothelzellen.The present invention relates to hemocompatible surfaces containing on and / or in the surface of the materials oligosaccharide, Polysaccharide and / or lipid portions of the glycoproteins, glycolipids and / or proteoglycans from the outer layer of blood cells and / or Mesothelial cells.

In einer weiteren Ausführungsvariante der vorliegenden Erfindung enthalten die hämokompatiblen Oberflächen auf und/oder in der Oberfläche der Werkstoffe Glykosphingolipide.In a further embodiment variant of the present invention contain the hemocompatible surfaces on and / or in the Surface of the materials glycosphingolipids.

Ferner können die hämokompatiblen Oberflächen der vorliegenden Erfindung als Oligosaccharid- oder Polysaccharid-Anteile der Proteoglykane Hyaluronsäuren, Chondroitinsulfate, Dermatansulfate, Heparansulfate, Keratansulfate oder Mischungen davon enthalten. In einer bevorzugten Ausführungsform der vorliegenden Erfindung enthalten die hämokompatiblen Oberflächen Heparansulfat der Erythrocyten-Plasmamembran tierischer und/oder menschlicher Herkunft. Furthermore, the hemocompatible surfaces of the present Invention as oligosaccharide or polysaccharide portions of the Proteoglycans hyaluronic acids, chondroitin sulfates, dermatan sulfates, Contain heparan sulfates, keratin sulfates or mixtures thereof. In a preferred embodiment of the present invention contain the hemocompatible surfaces heparan sulfate of the erythrocyte plasma membrane of animal and / or human origin.

Die erfindungsgemäß hämokompatiblen Oberflächen sind frei von Nebenwirkungen, wie sie z.B. durch chemisch oder pharmazeutisch aktive Beschichtungen bekannt sind.The hemocompatible surfaces according to the invention are free of Side effects such as through chemically or pharmaceutically active Coatings are known.

Bei den zuvor genannten Bestandteilen der Blut- und/oder Mesothelzellen handelt es sich um nicht-immunogene Zellbestandteile. Folglich zeichnen sich die erfindungsgemäß hämokompatiblen Oberflächen dadurch aus, daß sie außerdem nicht-immunogen sind. Das heißt, sie lösen bei den Patienten keine Immunreaktion aus, wodurch die Gefahr einer Abstoßung der hämokompatiblen Oberflächen minimiert ist.For the aforementioned components of the blood and / or mesothelial cells are non-immunogenic cell components. Therefore draw the hemocompatible surfaces according to the invention are characterized by that they are also non-immunogenic. That means, they solve with the Patients do not have an immune response, increasing the risk of rejection the hemocompatible surfaces is minimized.

Erfindungsgemäß sind die hämokompatiblen Oberfächen nicht-thrombogen und/oder nicht-immunogen.According to the invention, the hemocompatible surfaces are non-thrombogenic and / or non-immunogenic.

Ein weiterer Vorteil ist, daß aufgrund der erfindungsgemäßen festen Verankerung der nicht-thrombogenen Bestandteile der äußeren Schicht der Blut und/oder Mesothelzellen auf den Werkstoffen nahezu keine Degradation an den hämokompatiblen Oberflächen stattfindet. Eine Gefahr der Bildung von Embolien durch Thrombosen ist somit minimiert. Ferner erfolgt keine Anlagerung von Zellen, wie z.B. Thrombocyten auf den erfindungsgemäß hämokompatiblen Oberflächen. Dies minimiert ebenfalls die Gefahr von Thrombosen.Another advantage is that due to the solid according to the invention Anchoring the non-thrombogenic components of the outer layer the blood and / or mesothelial cells on the materials almost none Degradation takes place on the hemocompatible surfaces. A This minimizes the risk of embolism from thrombosis. Furthermore, there is no accumulation of cells, e.g. Platelets the hemocompatible surfaces according to the invention. This minimizes also the risk of thrombosis.

Gegenstand der vorliegenden Erfindung ist ferner ein Verfahren zur Herstellung der erfindungsgemäß hämokompatiblen Oberflächen, bei dem Glykophorine, Oligosaccharid-, Polysaccharid- und/oder Lipid-Anteile der Glykoproteine, Glykolipide und/oder Proteoglykane aus der äußeren Schicht von Blutzellen und/oder Mesothelzellen isoliert werden und diese Zellbestandteile durch physikalische oder chemische Bindung auf und/oder in die Oberfläche von Werkstoffen aus künstlichen und/oder natürlichen organischen und/oder anorganischen Verbindungen und/oder Mischungen davon und/oder Materialien, die bei invasiven Eingriffen mit Blut und/oder anderen Körperflüssigkeiten in Kontakt kommen und/oder tierischen Organen und/oder Organteilen auf- und/oder eingebracht werden.The present invention also relates to a method for Production of the hemocompatible surfaces according to the invention, in which Glycophorins, oligosaccharide, polysaccharide and / or lipid portions of the Glycoproteins, glycolipids and / or proteoglycans from the outside Layer of blood cells and / or mesothelial cells are isolated and this Cell components through physical or chemical bonding and / or in the surface of materials made of artificial and / or natural organic and / or inorganic compounds and / or Mixtures of these and / or materials used in invasive interventions Blood and / or other body fluids come into contact and / or animal organs and / or organ parts applied and / or introduced become.

Erfindungsgemäß werden die Bestandteile der äußeren Schicht von Blutzellen aus Vollblut und/oder aus daraus gewonnenen Zellfraktionen menschlicher oder tierischer Herkunft isoliert. D.h., daß die Zellbestandteile aus Erythrocyten, Leukocyten und/oder Thrombocyten oder Gemischen davon isoliert werden. Bevorzugt werden Gemische aus Erythrocyten und Leukocyten. Besonders bevorzugt werden Erythrocyten.According to the invention, the components of the outer layer of Blood cells from whole blood and / or from cell fractions obtained therefrom isolated from human or animal origin. This means that the Cell components from erythrocytes, leukocytes and / or thrombocytes or mixtures thereof can be isolated. Mixtures of are preferred Erythrocytes and leukocytes. Erythrocytes are particularly preferred.

Die Bestandteile aus der äußeren Schicht von Mesothelzellen werden erfindungsgemäß aus Omentum, Peritoneum und/oder inneren Organen isoliertThe components from the outer layer of mesothelial cells are according to the invention from omentum, peritoneum and / or internal organs isolated

Eine preiswerte und leicht zugängliche Quelle für diese Ausgangsmaterialien können beispielsweise Schlachtabfälle sein.An inexpensive and easily accessible source for this For example, raw materials can be slaughterhouse waste.

Die Isolierung der Bestandteile der äußeren Schicht der Blutzellen, Mesothelzellen oder des mesothelzellreichen Gewebes erfolgt dabei in an sich bekannter Weise. Beispielsweise sind hier folgende Verfahren oder deren Kombinationen denkbar: Zerkleinerung, Extraktion, Filtration, Fällung, Gelfiltration, Ionenaustauschchromatographie, Affinitätschromatographie, Elektrophorese, enzymatische oder chemische Abbauten, Trocknung, Auflösung, Dialyse, Ultrafiltration etc..Isolation of the components of the outer layer of blood cells, Mesothelial cells or tissue rich in mesothelial cells occurs in well-known way. For example, here are the following methods or their combinations are conceivable: shredding, extraction, filtration, Precipitation, gel filtration, ion exchange chromatography, Affinity chromatography, electrophoresis, enzymatic or chemical Degradation, drying, dissolution, dialysis, ultrafiltration etc.

Erfindungsgemäß wird zur Auf- und/oder Einbringung der Zellbestandteile auf und/oder in die Oberfläche der Werkstoffe eine chemische Immobilisierung, Photoimmobilisierung, Adhäsion, Trocknung oder eine Kombination davon durchgeführt. Dabei können kovalente, ionische, nebenvalente bzw. elektrostatische oder adhäsive Bindungen oder Kombinationen davon zwischen den Bestandteilen der äußeren Schicht der Zellen und den Oberflächen der Werkstoffe ausgebildet werden. Bevorzugt erfolgt die Auf- bzw. Einbringung der Bestandteile der äußeren Zellschicht auf/in die Oberfläche von Werkstoffen durch kovalente Bindungen.According to the invention for the application and / or introduction of the cell components a chemical on and / or in the surface of the materials Immobilization, photoimmobilization, adhesion, drying or a Combination of them performed. Covalent, ionic, incidental or electrostatic or adhesive bonds or Combinations thereof between the components of the outer layer of the cells and the surfaces of the materials. The outer components are preferably applied or introduced Cell layer on / in the surface of materials by covalent Bonds.

Ein besonderer Vorteil der vorliegenden Erfindung ist, daß das erfindungsgemäße Herstellungsverfahren enorme wirtschaftliche Verbesserungen gegenüber bisher bekannten Verfahren auf sich vereint und folglich die erfindungsgemäß hämokompatiblen Oberflächen zur Herstellung im großtechnischen Maßstab geeignet sind.
Dies basiert beispielsweise darauf, daß erfindungsgemäß Zellbestandteile und keine lebenden Zellen eingesetzt werden, daß keine körpereigenen (Endothel-) Zellen des Patienten verwendet werden müssen, daß das Ausgangsmaterial zur Isolierung dieser Zellbestandteile preiswert und in großen Mengen verfügbar ist (Schlachtabfälle), daß somit keine Zellkultivierung erforderlich ist, die sehr zeit- und kostenintensiv ist. Ein Vorteil der erfindungsgemäß hämokompatiblen Oberflächen ist femer, daß sie universell einsetzbar sind und nicht nur an den Einsatz für einen einzigen Patienten gebunden sind. Dies stellt vor allem bei Notoperationen einen lebenswichtigen Vorteil für die Patienten dar.A particular advantage of the present invention is that the manufacturing method according to the invention combines enormous economic improvements over previously known methods and consequently the hemocompatible surfaces according to the invention are suitable for manufacturing on an industrial scale.
This is based, for example, on the fact that, according to the invention, cell components and no living cells are used, that no endogenous (endothelial) cells of the patient have to be used, that the starting material for isolating these cell components is inexpensive and available in large quantities (slaughterhouse waste), so that none Cell cultivation is required, which is very time and cost intensive. Another advantage of the hemocompatible surfaces according to the invention is that they can be used universally and are not restricted to use for a single patient. This is a vital benefit for patients, especially in emergency operations.

Die Anwendungsgebiete der vorliegenden Erfindung sind weit gestreut. Die vorliegende Erfindung betrifft die Verwendung hämokompatibler Oberflächen in weiten Bereichen des Gesundheitssektors, in der Medizin, Zahnmedizin, Chirurgie oder Kosmetik und/oder in Bereichen, die bei invasiven Eingriffen mit Blut, Gewebe und/oder anderen Körperflüssigkeiten in Verbindung kommen.The fields of application of the present invention are widely spread. The present invention relates to the use of hemocompatible Surfaces in wide areas of the health sector, in medicine, Dentistry, surgery or cosmetics and / or in areas related to invasive procedures with blood, tissue and / or others Body fluids come into contact.

Im folgenden wird die Erfindung unter Bezugnahme auf die Beispiele näher erläutert, die jedoch nicht limitierend sind:The invention will now be described with reference to the examples explained in more detail, but which are not limiting:

1.) Isolierung von Erytrocytenplasmamembran Heparansulfat:1.) Isolation of erytrocyte plasma membrane heparan sulfate:

Ein Liter serumfrei gewaschene Erythrocyten werden in 1 Liter 0,154 molarem Phosphatpuffer pH 7 suspendiert und mit 1 U/ml Papain versetzt. Nach 2 Stunden Inkubation bei 56°C wird 20 Minuten bei 3000 g abzentrifugiert und der Überstand anschließend dekantiert. In diesem Überstand werden 100 ml DEAE-Sepharose CL-6B lonenaustauscher-Gel der Firma Pharmacia Biotech suspendiert. Das so beladene Gel wird noch dreimal in 0,1 molarer Kochsalz-Lösung gewaschen und in eine Chromatographiesäule gefüllt. Die Elution erfolgt mittels eines linearen Kochsalzgradienten im Bereich von 0,1 bis 0,8 mol/l über einem Gesamt-Elutionsvolumen von 2 Litern. Es werden 200 Fraktionen zu je 10 ml Volumen gesammelt. Die Fraktionen, die mit Dimethylmethylenblau (DMMB) der Firma Fluka nach der Methode beschrieben bei Chandrasekhar et al (Analytical Biochemistry, 161 (1987): 103-108) eine positive Farbreaktion ergeben, werden vereinigt. Die Lösung der gesammelten Fraktionen wird bei 26,7 hPa (20 Torr)und 40°C eingeengt und gegen Wasser dialysiert. Das Dialysat wird auf ein Volumen von 100 ml und eine Konzentration von 0,03 mol/l Natriumacetat, 0,073 mol/l Tris (Tris(hydroxymethyl)aminomethan der Firma Fluka) und pH 8.0 eingestellt, mit 1 U Chondroitinase ABC versetzt und 15 Stunden bei 37°C inkubiert. Nach Dialysieren gegen Wasser und Einengen unter Wasserstrahlvakuum wird die erhaltene Lösung erneut auf eine Säule mit 100 ml DEAE-Sepharose CL-6B der Firma Pharmacia Biotech. aufgetragen und wie zuvor beschrieben eluiert. Die DMMB-positiven Gradientenfraktionen werden dialysiert, unter Wasserstrahlvakuum auf ein Volumen von 1 ml eingeengt und auf einer Säule zur präparativen Gelfiltration (60 cm x 2 cm) unter Verwendung eines Sephacryl S-300 Gels (Pharmacia Biotech.) chromatographiert. Es werden 60 Fraktionen zu je 2 ml Volumen gesammelt, mit DMMB detektiert und die positiven Fraktionen vereinigt. Nach wiederholter Dialyse und Lyophilisation erhält man das aufgereinigte Erythrocytenplasmamembran-Heparansulfat.One liter of serum-free erythrocytes is 0.154 in 1 liter molar phosphate buffer pH 7 suspended and mixed with 1 U / ml papain. After 2 hours of incubation at 56 ° C for 20 minutes at 3000 g centrifuged and the supernatant then decanted. In this The supernatant is 100 ml of DEAE-Sepharose CL-6B ion exchange gel the company Pharmacia Biotech suspended. The gel so loaded will still be washed three times in 0.1 molar saline and into one Chromatography column filled. Elution is carried out using a linear Saline gradients in the range of 0.1 to 0.8 mol / l over a total elution volume of 2 liters. There are 200 fractions of 10 ml each Volume collected. The fractions containing dimethylmethylene blue (DMMB) from Fluka according to the method described in Chandrasekhar et al (Analytical Biochemistry, 161 (1987): 103-108) one positive color reaction are combined. The solution of Collected fractions are concentrated at 26.7 hPa (20 torr) and 40 ° C. and dialyzed against water. The dialysate is made up to a volume of 100 ml and a concentration of 0.03 mol / l sodium acetate, 0.073 mol / l Tris (Tris (hydroxymethyl) aminomethane from Fluka) and pH 8.0, mixed with 1 U chondroitinase ABC and incubated for 15 hours at 37 ° C. After dialyzing against water and concentrating under a water jet vacuum the solution obtained is again on a column with 100 ml of DEAE-Sepharose CL-6B from Pharmacia Biotech. applied and how previously eluted. The DMMB positive gradient fractions are dialyzed under a water jet vacuum to a volume of 1 ml concentrated and on a column for preparative gel filtration (60 cm x 2 cm) using a Sephacryl S-300 gel (Pharmacia Biotech.) Chromatograph. There are 60 fractions of 2 ml volume collected, detected with DMMB and the positive fractions pooled. After repeated dialysis and lyophilization, the purified is obtained Erythrocytenplasmamembran-heparan sulfate.

2.) Isolierung von Leukocytenoberflächen-Proteochondroitinsulfat:2.) Isolation of leukocyte surface proteochondroitin sulfate:

Ein Liter Citratblut wird 10 min in einer Zentrifuge mit Ausschwingrotor bei 3000 g zentrifugiert und das überstehende Plasma abgesaugt. Das Zellsediment wird mit 2 Litern auf 4°C gekühlter 1%iger Ammoniumoxalatlösung gemischt und 30 min bei derselben Temperatur inkubiert. Nach 5 minütiger Zentrifugation bei 500 g wird der rote Überstand verworfen und das Pellet in 2 Litern auf 4°C gekühlter 1%iger Ammoniumoxalatlösung suspendiert, 5 min bei 500 g zentrifugiert und der Waschvorgang, wie oben beschrieben noch zweimal wiederholt. Der nun farblose Überstand wird verworfen und das gewaschene Zellsediment (Ausbeute: 12 x 107 - 10 x 109 Zellen in 2 Litern Triton X-100 Puffer (0,5% Triton X-100, 10 mM Tris-HCI, 150 mM NaC1, pH 8) unter ständigem Rühren bei 25°C über 2 Stunden lysiert. Der Detergenzextrakt wird 60 min bei 10.000 g zentrifugiert, dekantiert und im Überstand werden im 10 ml DEAE Sephadex A50 lonenaustauscher Gel der Firma Pharmacia Biotech. suspendiert und sedimentiert. Das so beladene Gel wird noch dreimal in 0,1 molarer Kochsalz-Lösung gewaschen und in eine Chromatographiesäule gefüllt. Mit einem linearen Kochsalzgradienten von 0,1 bis 0,8 mol/l über ein Gesamt-Elutionvolumen von 2 Litern wird die Säule eluiert. Es werden 100 Fraktionen zu je 2 ml Volumen gesammelt und die Fraktionen, die mit Dimethylmethylenblau (DMMB der Firma Fluka) eine positive Farbreaktion ergeben, vereinigt. Die Lösung wird bei 26,7 hPa (20 Torr) und 40°C eingeengt und gegen Wasser dialysiert. Das Dialysat wird auf ein Volumen von 100 ml und eine Konzentration von 0,1 mmol/l Calciumacetat und 0,1 mol/l Natriumacetat eingestellt, mit Essigsäure auf pH 7 titriert, mit je 1 U Heparinase I, Heparinase II und Heparinase III versetzt und 15 Stunden bei 37°C inkubiert.
Nach Dialysieren gegen Wasser und Einengen unter Wasserstrahlvakuum wird die resultierende Lösung erneut auf eine Säule mit 10 ml DEAE Sephadex A50 der Firma Pharmacia Biotech. aufgetragen und wie oben beschrieben eluiert. Die DMMB-positiven Gradientenfraktionen werden dialysiert, unter Wasserstrahlvakuum auf ein Volumen von 1 ml eingeengt und auf einer Säule zur präparativen Gelfiltration (60 cm x 2 cm) unter Verwendung eines Sepharose Cl-4B Gels der Firma Pharmacia Biotech. chromatographiert. Es werden 60 Fraktionen zu je 2 ml Volumen gesammelt, mit DMMB detektiert und die positiven Fraktionen vereinigt. Nach wiederholter Dialyse und Lyophilisation erhält man das aufgereinigte Leukocytenoberflächen-Proteochondroitinsulfat.One liter of citrate blood is centrifuged at 3000 g in a centrifuge with a swing-out rotor and the supernatant plasma is suctioned off. The cell sediment is mixed with 2 liters of 1% ammonium oxalate solution cooled to 4 ° C. and incubated for 30 minutes at the same temperature. After centrifugation at 500 g for 5 minutes, the red supernatant is discarded and the pellet is suspended in 2 liters of 1% ammonium oxalate solution cooled to 4 ° C., centrifuged at 500 g for 5 min and the washing process is repeated twice as described above. The now colorless supernatant is discarded and the washed cell sediment (yield: 12 × 10 7-10 × 10 9 cells in 2 liters of Triton X-100 buffer (0.5% Triton X-100, 10 mM Tris-HCl, 150 mM NaCl , pH 8) with constant stirring for 2 hours at 25 ° C. The detergent extract is centrifuged at 10,000 g for 60 min, decanted and suspended in the supernatant and sedimented in 10 ml DEAE Sephadex A50 ion exchange gel from Pharmacia Biotech loaded gel is washed three more times in 0.1 molar sodium chloride solution and filled into a chromatography column, the column being eluted with a linear sodium chloride gradient of 0.1 to 0.8 mol / l over a total elution volume of 2 liters 100 fractions of 2 ml volume were collected and the fractions which gave a positive color reaction with dimethylmethylene blue (DMMB from Fluka) were combined The solution was concentrated at 26.7 hPa (20 torr) and 40 ° C. and dialyzed against water. The dialysate will adjusted to a volume of 100 ml and a concentration of 0.1 mmol / l calcium acetate and 0.1 mol / l sodium acetate, titrated to pH 7 with acetic acid, mixed with 1 U heparinase I, heparinase II and heparinase III and 15 hours incubated at 37 ° C.
After dialyzing against water and concentrating under a water jet vacuum, the resulting solution is again placed on a column with 10 ml DEAE Sephadex A50 from Pharmacia Biotech. applied and eluted as described above. The DMMB-positive gradient fractions are dialyzed, concentrated to a volume of 1 ml under water jet vacuum and on a column for preparative gel filtration (60 cm × 2 cm) using a Sepharose Cl-4B gel from Pharmacia Biotech. Chromatograph. 60 fractions of 2 ml volume are collected, detected with DMMB and the positive fractions are combined. After repeated dialysis and lyophilization, the purified leukocyte surface proteochondroitin sulfate is obtained.

3.) Isolierung von Heparansulfat/Chondroitinsulfat-Gemisch aus Omentum:3.) Isolation of heparan sulfate / chondroitin sulfate mixture from omentum:

Ein Kilogramm frisches Rinderomentum wird mit 0,9%iger NaCI-Lösung gewaschen, gefriergetrocknet, gemahlen und mit 1 Liter Aceton durch Rühren über Nacht bei Raumtemperatur entfettet. Nach dem Abfiltrieren und Trocknen wird das resultierende Pulver in 6 molarer Harnstoff-Lösung suspendiert und über Nacht bei Raumtemperatur gerührt. Nach Zentrifugation bei 3000 g für 1 Stunde wird der schleimige Überstand dekantiert, auf 4°C gekühlt, mit dem gleichen Volumen 4°C kalter 1 molarer NaOH gemischt und 15 Stunden bei 4°C inkubiert. Danach wird mit verdünnter HCI neutralisiert, gegen Wasser dialysiert, 1 Stunde bei 3000 g zentrifugiert und der Überstand dekantiert. Im Überstand werden 100 ml DEAE-Sepharose CL-6B Ionenaustauscher-Gel der Firma Pharmacia Biotech. suspendiert und sedimentiert. Das so beladene Gel wird noch dreimal in 0,1 molarer Kochsalz-Lösung gewaschen und in eine Chromalographiesäule gefüllt. Mit einem linearen Kochsalzgradienten von 0,1 bis 0,8 mol/l über einem Gesamt-Elutionsvolumen von 2 Litern wird die Säule eluiert. Es werden 200 Fraktionen zu je 10 ml Volumen gesammelt und die Fraktionen, die mit Dimethylmethylenblau (DMMB) eine positive Farbreaktion ergeben, vereinigt. Die Lösung wird bei 26,7 hPa (20 Torr) und 40°C eingeengt und gegen Wasser dialysiert. Unter Wasserstrahlvakuum wird erneut auf ein Volumen von 5 ml eingeengt und auf einer Säule zur präparativen Gelfiltration (60 cm x 5 cm) unter Verwendung eines Sephacryl S-300 Gels der Firma Pharmacia Biotech. chromatographlert. Es werden 60 Fraktionen zu je 10 ml Volumen gesammelt, mit DMMB detektiert und die positiven Fraktionen vereinigt. Nach wiederholter Dialyse und Lyophilisation erhält man das aufgereinigte Mesothelzelloberflächen-Glykosaminoglykan-Gemisch. A kilogram of fresh beef momentum is made with 0.9% NaCI solution washed, freeze-dried, ground and mixed with 1 liter of acetone Degreased stirring overnight at room temperature. After filtering off and drying the resulting powder in 6 molar urea solution suspended and stirred overnight at room temperature. To Centrifugation at 3000 g for 1 hour becomes the slimy supernatant decanted, chilled to 4 ° C, with the same volume 4 ° C cold 1 molar NaOH mixed and incubated for 15 hours at 4 ° C. After that neutralized with dilute HCI, dialyzed against water, at 1 hour Centrifuged 3000 g and the supernatant decanted. Be in the supernatant 100 ml DEAE-Sepharose CL-6B ion exchange gel from the company Pharmacia Biotech. suspended and sedimented. The gel so loaded is washed three more times in 0.1 molar sodium chloride solution and in a Chromalography column filled. With a linear saline gradient of 0.1 to 0.8 mol / l over a total elution volume of 2 liters Column eluted. 200 fractions of 10 ml volume are collected and the fractions containing dimethylmethylene blue (DMMB) are positive Color reaction result, united. The solution is at 26.7 hPa (20 torr) and 40 ° C and dialyzed against water. Under Water jet vacuum is again concentrated to a volume of 5 ml and on a column for preparative gel filtration (60 cm x 5 cm) under Use of a Sephacryl S-300 gel from Pharmacia Biotech. chromatographlert. There are 60 fractions of 10 ml volume collected, detected with DMMB and the positive fractions pooled. After repeated dialysis and lyophilization, the purified is obtained Mesothelzelloberflächen-glycosaminoglycan mixture.

5.) Immobilisierung von Mesothelzelloberflächen Chondroitinsulfat mit (N-Cyclohexyl-N'-2-5.) Immobilization of mesothelial cell surfaces Chondroitin sulfate with (N-cyclohexyl-N'-2- morpholinoethyl)carbodiimidmethyl p-Toluolsulfonat (CME-CDI) auf funktionalisierte Zelluloseoberflächen:morpholinoethyl) carbodiimide methyl p-toluenesulfonate (CME-CDI) Functionalized cellulose surfaces:

100 mg Zellulosemembran werden in eine 2 prozentige Lösung von 3-Aminopropyl-triethoxysilan in Ethanol/Wasser (50:50) gegeben und 24 Stunden bei 45°C gerührt. Danach werden die Membranen mit viel Wasser gewaschen und getrocknet. Die so behandelten Membranen werden in eine Lösung von 1 mg Mesothelzelloberflächen Chondroitinsulfat in 80 ml 0,1 molarer 2-(N-Morpholino)ethansulfonsäure-Puffer pH 4,75 getaucht. Über einen Zeitraum von 6 Stunden werden bei 4°C 200 mg (N-Cyclohexyl-N'-2-morpholinoethyl)carbodiimidmethyl p-Toluolsulfonat (CME-CDI) der Firma Sigma in 10 mg Portionen zugegeben und über Nacht bei 4°C weitergerührt. Danach wird 2 Stunden in 4 molarer NaCl-Lösung gerührt, mit viel Wasser gespült und an der Luft getrocknet.100 mg cellulose membrane are in a 2 percent solution of 3-aminopropyl-triethoxysilane placed in ethanol / water (50:50) and 24 Stirred at 45 ° C for hours. After that, the membranes with a lot Washed water and dried. The membranes treated in this way are in a solution of 1 mg mesothelial cell surfaces Chondroitin sulfate in 80 ml 0.1 molar 2- (N-morpholino) ethanesulfonic acid buffer pH 4.75 submerged. Over a period of 6 hours 4 ° C 200 mg (N-cyclohexyl-N'-2-morpholinoethyl) carbodiimide methyl p-toluenesulfonate (CME-CDI) from Sigma added in 10 mg portions and stirred overnight at 4 ° C. Then 2 hours in 4 molar Stirred NaCl solution, rinsed with plenty of water and air dried.

6.) CNCI Immobilisierung von Sphingoglycolipid auf Glas:6.) CNCI immobilization of sphingoglycolipid on glass:

Ein Glas, beispielsweise ein Deckgläschen für die Mikroskopie, wird 6 Stunden in 5 ml Chromschwefelsäure gerührt. Dann wird mit viel Wasser gewaschen, luftgetrocknet und in 15 ml Dioxan auf 50°C erhitzt. Anschließend werden 2,5 ml einer 2 molaren N,N'-Diisopropylethylamin-Lösung in Dioxan zugegeben und 30 min gerührt. Dann werden 2,5 ml einer 1 molaren CNCI-Lösung in Dioxan zugeführt und für weitere 2 Stunden gerührt. Anschließend wird zuerst mit Dioxan gewaschen, dann mit Dioxan/Wasser und schließlich mit reinem Wasser gewaschen. Das so modifizierte Glas wird in 20 ml einer Lösung aus 1 mol/l Ethylendiamin und 0,1 mol/l NaHCO3 gegeben, dann auf 50°C erwärmt und 72 Stunden bei dieser Temperatur gerührt. Anschließend werden 0,1 mg Sphingoglycolipid aus humanen Erythrocyten in 20 ml 0,1 molarer NaHCO3 gelöst und zusammen mit dem substituierten Glas 110 Stunden bei 60°C gerührt. Dann werden 2,5 ml Ethanolamin hinzu gegeben und weitere 30 Minuten gerührt. Das beschichtete Glas wird mit 4 molarer NaCl-Lösung und anschließend mit viel Wasser gewaschen und an der Luft getrocknet.A glass, for example a cover slip for microscopy, is stirred in 5 ml of chromic sulfuric acid for 6 hours. Then it is washed with plenty of water, air-dried and heated to 50 ° C. in 15 ml of dioxane. Then 2.5 ml of a 2 molar N, N'-diisopropylethylamine solution in dioxane are added and the mixture is stirred for 30 min. Then 2.5 ml of a 1 molar CNCI solution in dioxane are added and the mixture is stirred for a further 2 hours. It is then washed first with dioxane, then with dioxane / water and finally with pure water. The glass modified in this way is placed in 20 ml of a solution of 1 mol / l of ethylenediamine and 0.1 mol / l of NaHCO 3 , then heated to 50 ° C. and stirred at this temperature for 72 hours. Then 0.1 mg sphingoglycolipid from human erythrocytes is dissolved in 20 ml 0.1 molar NaHCO 3 and stirred together with the substituted glass at 60 ° C for 110 hours. Then 2.5 ml of ethanolamine are added and the mixture is stirred for a further 30 minutes. The coated glass is washed with 4 molar NaCl solution and then with plenty of water and air dried.

7.) Immobilisierung von Erythrocytenplasmamembran-Heparansulfat auf die Oxidschicht von Nickel, Titan, Aluminium oder ähnlichen Metallen:7.) Immobilization of erythrocyte plasma membrane heparan sulfate on the oxide layer of nickel, titanium, aluminum or similar metals:

Das Metallwerkstück wird vier Stunden im Ultraschallbad mit heißem Wasser gereinigt, mit Aceton gewaschen und eine Stunde in einem Soxhlet-Extraktor mit Chloroform entfettet. Das so gereinigte Werkstück wird getrocknet und 2-15 min unter Rühren in eine 0,01 -0,1 molare Lösung von ω-Hexadecenyltrichlorsilan in Bicyclohexyl getaucht, zweimal mit Chloroform und Wasser gewaschen und 15 min im Soxhlet-Extraktor mit Chloroform extrahiert. Das Werkstück wird bei O°C 45 min in eine Lösung von 2 ml Aceton und 100 mg KmnO4 in 18 ml Wasser getaucht und durch die einen CO2-Strom geleitet. Danach wird es für 15 Sekunden in eine 20%ige Lösung von Natriumbisulfit in Wasser getaucht, mit Wasser gewaschen und getrocknet.
Das Werkstück wird über Nacht in einer Lösung von 29,25 g Paratoluylsulfonylchlorid in 900 ml Aceton und 180 ml Pyridin bei 40°C gerührt. Anschließend wird das Werkstück mit Wasser und Methanol gewaschen und 40 Stunden lang bei 60°C in einer Lösung von 1 mmol/l Diaminododekan in 1 Liter Dimethylformamid gerührt. Danach wird das Werkstück sukzessive mit Wasser, 1 mol/l Sodalösung, 1 mmol/l Salzsäure und Wasser gewaschen. Das so vorbereitete Werkstück wird für 90 Minuten in einer Borat-Pufferlösung (Natriumtetraborat 0,065 mol/l, pH 9,5) gerührt. Schließlich wird in einer Lösung aus 0,3 g 4-Azido-1-Fluoro-2-Nitrobenzol in einem Liter Ethanol über Nacht bei 37°C gerührt. 0,5 g Erythrocytenplasmamembran-Heparansulfat werden in einem Liter 0,1 molaren 2-(N-Morpholino)ethansulfonsäure-(MES)-Puffer pH 4,75 gelöst und mit dem Werkstück bei 4°C für 48 Stunden gerührt. Das Erythrocytenplasmamembran-Heparansulfat wird kovalent immobilisiert 10 minütige Belichtung mit einer Hochdruck-Quecksilberlampe. Nach Waschen mit 4 molarer Kochsalzlösung für 40 Minuten wird das Werkstück mit Wasser gewaschen und anschließend getrocknet.The metal workpiece is cleaned with hot water in an ultrasound bath for four hours, washed with acetone and degreased with chloroform in an Soxhlet extractor for one hour. The workpiece cleaned in this way is dried and immersed in a 0.01-0.1 molar solution of ω-hexadecenyltrichlorosilane in bicyclohexyl with stirring for 2-15 minutes, washed twice with chloroform and water and extracted with chloroform in a Soxhlet extractor for 15 minutes. The workpiece is immersed in a solution of 2 ml of acetone and 100 mg of KmnO 4 in 18 ml of water at 0 ° C. for 45 minutes and passed through a CO 2 stream. Then it is immersed in a 20% solution of sodium bisulfite in water for 15 seconds, washed with water and dried.
The workpiece is stirred at 40 ° C. overnight in a solution of 29.25 g of paratoluylsulfonyl chloride in 900 ml of acetone and 180 ml of pyridine. The workpiece is then washed with water and methanol and stirred for 40 hours at 60 ° C. in a solution of 1 mmol / l diaminododecane in 1 liter of dimethylformamide. The workpiece is then washed successively with water, 1 mol / l sodium carbonate solution, 1 mmol / l hydrochloric acid and water. The workpiece prepared in this way is stirred for 90 minutes in a borate buffer solution (sodium tetraborate 0.065 mol / l, pH 9.5). Finally, it is stirred in a solution of 0.3 g of 4-azido-1-fluoro-2-nitrobenzene in one liter of ethanol at 37 ° C. overnight. 0.5 g of erythrocyte plasma membrane heparan sulfate is dissolved in one liter of 0.1 molar 2- (N-morpholino) ethanesulfonic acid (MES) buffer pH 4.75 and stirred with the workpiece at 4 ° C. for 48 hours. The erythrocyte plasma membrane heparan sulfate is covalently immobilized for 10 minutes by exposure to a high-pressure mercury lamp. After washing with 4 molar saline for 40 minutes, the workpiece is washed with water and then dried.

8.) Photochemische Immobilisierung von Leukocytenplasmamembran-Chondroitinsulfat auf Zellulose:8.) Photochemical immobilization of Leukocyte plasma membrane chondroitin sulfate on cellulose:

3 g Zellulosemembran werden in 4 molarer NaOH 2 Stunden quellen gelassen, dreimal mit Wasser gewaschen, einmal mit Wasser/Aceton und einmal mit Aceton gewaschen. Die so aktivierte Zellulose wird über Nacht in einer Lösung von 29,25 g Paratoluylsulfonylchlorid in 900 ml Aceton und 180 ml Pyridin bei 40°C gerührt. Anschließend wird die Zellulosemembran mit Wasser und Methanol gewaschen. Die resultierende veresterte Zellulosemembran wird nun über 40 Stunden bei 60°C in einer Lösung von 1 mmol/l Diaminododekan in 1 Liter Dimethylformamid gerührt. Danach wird die Membran sukzessive mit Wasser, 1 mol/l Sodalösung, 1 mmol/l Salzsäure und Wasser gewaschen. Die so erhaltene Aminozellulose wird für 90 Minuten in einer Borat-Pufferlösung (Natriumtetraborat 0,065 molar, pH 9,5) gerührt. Schließlich wird die Membran in einer Lösung aus 0,3 g 4-Azido-1-Fluoro-2-Nitrobenzol in einem Liter Ethanol aber Nacht bei 37°C gerührt. 0,5 g Leukocytenoberflächen-Chondroitinsulfat werden in einem Liter 0,1 molaren 2-(N-Morpholino)ethansulfonsäure-Puffer pH 4,75 gelöst und mit 2,5 g der wie oben beschrieben hergestellten Azido-Zellulose bei 4°C für 48 Stunden gerührt. Das Leukocytenoberflächen-Chondroitinsulfat wird kovalent immobilisiert durch 10 minütige Belichtung mit einer Hochdruck-Quecksilberlampe. Nach Waschen mit 4 molarer Kochsalzlösung für 40 Minuten und Wasser wird die Zellulosemembran getrocknet.3 g of cellulose membrane will swell in 4 molar NaOH for 2 hours left, washed three times with water, once with water / acetone and washed once with acetone. The cellulose activated in this way becomes overnight in a solution of 29.25 g paratoluylsulfonyl chloride in 900 ml acetone and 180 ml of pyridine at 40 ° C stirred. Then the Cellulose membrane washed with water and methanol. The resulting esterified cellulose membrane is now over 40 hours 60 ° C in a solution of 1 mmol / l diaminododecane in 1 liter Dimethylformamide stirred. Then the membrane is gradually added Washed water, 1 mol / l soda solution, 1 mmol / l hydrochloric acid and water. The amino cellulose thus obtained is soaked in a borate buffer solution for 90 minutes (Sodium tetraborate 0.065 molar, pH 9.5). Finally the membrane is in a solution of 0.3 g of 4-azido-1-fluoro-2-nitrobenzene stirred in a liter of ethanol at 37 ° C overnight. 0.5 g Leukocyte surface chondroitin sulfate is 0.1 in one liter molar 2- (N-morpholino) ethanesulfonic acid buffer pH 4.75 dissolved and with 2.5 g of the azido cellulose prepared as described above at 4 ° C for Stirred for 48 hours. The leukocyte surface chondroitin sulfate is covalently immobilized by exposure to a high-pressure mercury lamp for 10 minutes. After washing with 4 molar saline for 40 Minutes and water, the cellulose membrane is dried.

9.) Immobilisierung von Glycophorin A mit Glutardialdehyd auf Silikon:9.) Immobilization of glycophorin A with glutardialdehyde Silicone:

1 g Silikonfolie wird mit 20 ml Wasser und 2 ml 3-Aminopropyltriethoxysilan versetzt und der pH-Wert auf 3,5 eingestellt. Dann wird für 2 Stunden auf 75°C erhitzt, mit Wasser gewaschen und getrocknet. Das so erhaltene Aminogruppen-haltige Silikon wird mit einer 2,5 prozentigen Lösung von Glutardialdehyd in 0,05 molarem Natriumphosphatpuffer versetzt und auf pH 7 eingestellt. Nach 60 Minuten Rühren bei Raumtemperatur wird das so hergestellte aktivierte Silikon mit einer 0,1%igen Lösung von Glycophorin A (Sigma) unter Rühren 2-4 Stunden umgesetzt und mit Wasser gewaschen.1 g of silicone foil is mixed with 20 ml of water and 2 ml of 3-aminopropyltriethoxysilane added and the pH adjusted to 3.5. Then it is heated to 75 ° C. for 2 hours, washed with water and dried. The amino group-containing silicone thus obtained is with a 2.5 percent solution of glutardialdehyde in 0.05 molar Sodium phosphate buffer added and adjusted to pH 7. After 60 minutes The activated silicone thus produced is stirred with at room temperature a 0.1% solution of glycophorin A (Sigma) with stirring 2-4 Implemented for hours and washed with water.

10.) Immobilisierung von Erythrocytenplasmamembran-Heparansulfat auf Polyvinylchlorid (PVC):10.) Immobilization of erythrocyte plasma membrane heparan sulfate on polyvinyl chloride (PVC):

0,5 g Eisen-II-sulfat, 100 µl konzentrierte Schwefelsäure und 2 ml Methacrvlsäure werden in 250 ml Wasser gelöst. Zu dieser Lösung werden 125 mg Natriumdisulfit sowie 125 mg Kaliumperoxodisulfat zugegeben. Anschließend wird diese Lösung zwei Stunden bei Raumtemperatur durch einen ringförmigen 1 m langen PVC-Schlauch von 3 mm Innendurchmesser gepumpt. Die dabei ablaufende Pfropfpolymerisation wird durch Zugabe von 100 mg Hvdrochinon abgebrochen. Dann wird der Schlauch gründlich mit Wasser gewaschen. Eine auf 4°C gekühlte Lösung von 250 mg CME-CDI (N-Cyclohexyl-N'-2-morpholinoethyl)carbodiimidmethyl p-Toluolsulfonat in 250 ml 0,1 molarem 2-(N-Morpholino)ethansulfonsäure-Puffer pH 4,75 wird bei 4°C 30 min im Kreis durch den Schlauch gepumpt. Der so aktivierte Schlauch wird mit 0,1 molarem 2-(N-Morpholino)ethansulfonsäure-Puffer pH 4,75 gewaschen. Dann wird eine Lösung von 1 mg Erythrocytenplasmamembran-Heparansulfat in 0,1 molarem 2-(N-Morpholino)ethansulfonsäure-Puffer pH 4,75 bei 4°C 15 Stunden lang im Kreis durch den Schlauch gepumpt. Abschließend wird der Schlauch mit 4 molarer Kochsalzlösung und dann mit Wasser gespült.0.5 g iron (II) sulfate, 100 µl concentrated sulfuric acid and 2 ml Methacrylic acid are dissolved in 250 ml of water. About this solution 125 mg sodium disulfite and 125 mg potassium peroxodisulfate added. Then this solution is two hours Room temperature through an annular 1 m long PVC hose from 3 mm inner diameter pumped. The expiring Graft polymerization is carried out by adding 100 mg of hvdroquinone canceled. Then the hose is washed thoroughly with water. A solution of 250 mg of CME-CDI (N-cyclohexyl-N'-2-morpholinoethyl) carbodiimide methyl cooled to 4 ° C. p-toluenesulfonate in 250 ml 0.1 molar 2- (N-Morpholino) ethanesulfonic acid buffer pH 4.75 is at 4 ° C in 30 min Circle pumped through the hose. The hose activated in this way is included 0.1 molar 2- (N-morpholino) ethanesulfonic acid buffer pH 4.75 washed. Then a solution of 1 mg Erythrocyte plasma membrane heparan sulfate in 0.1 molar 2- (N-morpholino) ethanesulfonic acid buffer pH 4.75 at 4 ° C for 15 hours in Circle pumped through the hose. Finally, the hose with 4 molar saline and then rinsed with water.

Claims (12)

  1. Hemocompatible surfaces, characterized in that they contain as materials artificial and/or natural organic and/or inorganic compounds and/or mixtures thereof and/or materials having contact with blood and/or other body fluids in invasive operations and/or animal organs and/or organ parts, and oligosaccharide, polysaccharide and/or lipid portions of the glycoproteins, glycolipids and/or proteoglycans from the outer layer of blood cells and/or mesothelial cells are applied and/or incorporated onto and/or into the surfaces of said materials.
  2. The hemocompatible surfaces according to claim 1, characterized in that they are non-thrombogenic and/or non-immunogenic.
  3. The hemocompatible surfaces according to one of claims 1 or 2, containing glycophorins on and/or in the surfaces of the materials.
  4. The hemocompatible surfaces according to one of claims 1 - 3, containing glycosphingolipids on and/or in the surfaces of the materials.
  5. The hemocompatible surfaces according to one of claims 1 - 4, containing on and/or in the surfaces of the materials as the oligosaccharide and/or polysaccharide portions of the proteoglycans hyaluronic acids, chondroitin sulfates, dermatan sulfates, heparan sulfates, keratan sulfates or mixtures thereof.
  6. The hemocompatible surfaces according to one of claims 1 - 5, containing on and/or in the surfaces of the materials heparan sulfate of the erythrocyte plasma membrane of animal and/or human origin.
  7. The hemocompatible surfaces according to one of claims 1 - 6, containing as the materials high-molecular organic compounds and/or metals, metal oxides, alloys, ceramics, glasses, minerals and/or mixtures of the materials mentioned before.
  8. A process for making hemocompatible surfaces, characterized in that
    a) glycophorins and/or oligosaccharide, polysaccharide and/or lipid portions of the glycoproteins, glycolipids and/or proteoglycans are isolated from the outer layer of blood cells and/or mesothelial cells, and
    b) said cell constituents are applied and/or incorporated onto and/or into the surfaces of materials of artificial and/or natural organic and/or inorganic compounds and/or mixtures thereof and/or materials having contact with blood and/or other body fluids in invasive operations and/or animal organs and/or organ parts by physical or chemical bonding.
  9. The process according to claim 8, characterized in that the constituents from the outer layer of blood cells are isolated from whole blood and/or from cell fractions obtained therefrom of human or animal origin.
  10. The process according to one of claims 8 or 9, characterized in that cell constituents are isolated from erythrocytes, leucocytes and/or thrombocytes and/or mixtures thereof.
  11. The process according to one of claims 8 - 10, characterized in that constituents from the outer layer of mesothelial cells are isolated from omentum, peritoneum and/or inner organs.
  12. The process according to one of claims 8 - 11, characterized in that a chemical immobilization, photoimmobilization, adhesion, drying process or a combination thereof is carried out for applying and/or incorporating the cell constituents onto and/or into the surfaces of the materials.
EP00922483A 1999-02-26 2000-02-24 Hemocompatible surfaces and method for producing same Expired - Lifetime EP1152778B1 (en)

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DE19908318A DE19908318A1 (en) 1999-02-26 1999-02-26 Hemocompatible surfaces and methods of making them
DE19908318 1999-02-26
PCT/EP2000/001497 WO2000050106A2 (en) 1999-02-26 2000-02-24 Hemocompatible surfaces and method for producing same

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008086794A2 (en) 2007-01-21 2008-07-24 Hemoteq Ag Medical product for treating stenosis of body passages and for preventing threatening restenosis
EP2386322A2 (en) 2006-07-03 2011-11-16 Hemoteq AG Production, method and use of medical products which release agents for opening blood vessels on a permanent basis

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1568166A (en) * 2001-10-15 2005-01-19 荷姆泰克股份有限公司 Coating of stents for preventing restenosis
ES2321082T3 (en) 2002-05-09 2009-06-02 Hemoteq Ag MEDICAL PRODUCTS THAT INCLUDE A HEMOCOMPATIBLE COATING, PRODUCTION AND USE OF THE SAME.
DE10329296B4 (en) * 2003-06-24 2007-07-12 Leibniz-Institut Für Polymerforschung Dresden E.V. Coating system for biomaterials
DE102005040211B4 (en) 2005-08-16 2010-02-11 Maquet Cardiopulmonary Ag Use of nonionic esters in a coating for blood contacting surfaces and medical device
US9192697B2 (en) 2007-07-03 2015-11-24 Hemoteq Ag Balloon catheter for treating stenosis of body passages and for preventing threatening restenosis
ES2550634T3 (en) 2009-07-10 2015-11-11 Boston Scientific Scimed, Inc. Use of nanocrystals for a drug delivery balloon
JP5933434B2 (en) 2009-07-17 2016-06-08 ボストン サイエンティフィック サイムド，インコーポレイテッドＢｏｓｔｏｎ Ｓｃｉｅｎｔｉｆｉｃ Ｓｃｉｍｅｄ，Ｉｎｃ． Method for producing drug delivery balloon
WO2012031236A1 (en) 2010-09-02 2012-03-08 Boston Scientific Scimed, Inc. Coating process for drug delivery balloons using heat-induced rewrap memory
US8669360B2 (en) 2011-08-05 2014-03-11 Boston Scientific Scimed, Inc. Methods of converting amorphous drug substance into crystalline form
US9056152B2 (en) 2011-08-25 2015-06-16 Boston Scientific Scimed, Inc. Medical device with crystalline drug coating
CN102501344B (en) * 2011-09-29 2014-07-16 华东交通大学 Method for constructing hemocompatible material surface with bionic periodic structure
ES2764146T3 (en) 2013-02-08 2020-06-02 Acell Inc Methods for making bioactive gels from extracellular matrix material

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4350629A (en) * 1981-07-29 1982-09-21 Massachusetts Institute Of Technology Procedures for preparing composite materials from collagen and glycosaminoglycan
DE3639561A1 (en) * 1986-11-20 1988-06-01 Baumann Hanno METHOD FOR PRODUCING NON-THROMBOGEN SUBSTRATES
US5236835A (en) * 1988-10-05 1993-08-17 Hapgood, C.V. Electro insertion of proteins into red cell membranes
US5043164A (en) * 1989-01-17 1991-08-27 The University Of Tennessee Research Corporation Blood-stable, cholesterol-free liposomes
GB9116036D0 (en) * 1991-07-25 1991-09-11 Univ Leicester Preparing grafts for implantation
DE4402064A1 (en) * 1994-01-25 1995-07-27 Kohlensaeurewerk Deutschland Process for controlling pests in bulk material
US5643712A (en) * 1994-05-20 1997-07-01 Brasile; Lauren Method for treating and rendering grafts nonthrombogenic and substantially nonimmunogenic using an extracellular matrix coating
DE19505070C2 (en) * 1995-02-15 1997-03-27 Axel Prof Dr Med Haverich Artificial vascular systems and processes for their manufacture
DE19648876C2 (en) * 1996-11-16 1999-10-07 Will Minuth Method of making a natural implant
DE19700078A1 (en) * 1997-01-03 1998-07-09 Huels Chemische Werke Ag Production of polymeric materials with cell proliferation-promoting properties
DE19706667A1 (en) * 1997-02-20 1998-08-27 Merck Patent Gmbh Bone replacement material with a surface covering with peptides with RGD amino acid sequence
DE19724869C2 (en) * 1997-06-12 1999-05-12 Henkel Kgaa Use of citosan derivatives for surface coating

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2386322A2 (en) 2006-07-03 2011-11-16 Hemoteq AG Production, method and use of medical products which release agents for opening blood vessels on a permanent basis
WO2008086794A2 (en) 2007-01-21 2008-07-24 Hemoteq Ag Medical product for treating stenosis of body passages and for preventing threatening restenosis
EP2269664A2 (en) 2007-01-21 2011-01-05 Hemoteq AG Medical product for treating closures of bodily passages and preventing reclosures
EP2491962A1 (en) 2007-01-21 2012-08-29 Hemoteq AG Medical product for treating closures of bodily passages and preventing reclosures

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KR100503913B1 (en) 2005-07-26
DE19908318A1 (en) 2000-08-31
BR0008968A (en) 2001-11-27
IL144912A0 (en) 2002-06-30
CN1198660C (en) 2005-04-27
CA2363119C (en) 2007-04-10
ATE250437T1 (en) 2003-10-15
AU758873B2 (en) 2003-04-03
WO2000050106A3 (en) 2001-04-26
IL144912A (en) 2009-09-22
EP1152778A1 (en) 2001-11-14
CA2363119A1 (en) 2000-08-31
JP3597474B2 (en) 2004-12-08
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CN1341033A (en) 2002-03-20
AU4287100A (en) 2000-09-14

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